Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state.
Abstract
A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity. Study holds ProTherm entries: 17394, 17395, 17396 Extra Details: N-terminal 17-residue beta-hairpin. domain(MQIFVKTLTGKTITLEV);. dG_H2O was obtained with the mean m-value of 10.28 kJ/mol/M; wild type protein was of pseudo type, F45W nonnative local interactions; peptide fragment; beta-strand alignment; turn mutations; secondary structure
Submission Details
ID: kgGLnzaY3
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:49 p.m.
Version: 1
Publication Details
Platt GW;Simpson SA;Layfield R;Searle MS,Biochemistry (2003) Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state. PMID:14622023Additional Information
Study Summary
| Number of data points | 9 |
| Proteins | E3 ubiquitin-protein ligase RSP5 ; Ubiquitin-binding protein CUE2 |
| Unique complexes | 1 |
| Assays/Quantities/Protocols | Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O |
| Libraries | Mutations for sequence A:NDDHESKLSILMDMFPAISKSKLQVHLLENNNDLDLTIGLLLKENDDKS/B:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
|---|
Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 100.0 | E3 ubiquitin-protein ligase RSP5 | P39940 | RSP5_YEAST | |
| 100.0 | A | Ubiquitin-binding protein CUE2 | P36075 | CUE2_YEAST |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CH06 | RL401_SCHPO |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CH07 | RL402_SCHPO |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CH08 | RL40A_YEAST |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CH09 | RL40B_YEAST |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P40909 | RL40_CRYNJ |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CG73 | UBI1P_CANAX |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CG74 | UBI4P_CANAX |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CG75 | UBI4P_KLULA |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CG72 | UBI4P_SCHPO |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0CG63 | UBI4P_YEAST |
| 98.7 | B | Ubiquitin-binding protein CUE2 | P19848 | UBIQ_COPCO |
| 98.7 | B | Ubiquitin-binding protein CUE2 | P0C224 | RL40_NEUCR |
| 98.7 | B | Ubiquitin-binding protein CUE2 | P0CG70 | UBI4P_NEUCR |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P59271 | R27AA_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | B9DHA6 | RL40A_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH34 | RL40A_ORYSJ |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q42202 | RL40B_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH35 | RL40B_ORYSJ |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P51423 | RL40_BRARP |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P49636 | RL40_NICSY |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P62980 | RS27A_SOLLC |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P62981 | RS27A_SOLTU |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q9SHE7 | RUB1_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0C073 | RUB1_DESAN |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0C030 | RUB1_ORYSJ |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q8RUC6 | RUB2_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0C031 | RUB2_ORYSJ |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CG85 | UBI1P_NICSY |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH04 | UBI1P_PETCR |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH05 | UBI2P_PETCR |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CG84 | UBI4P_NICSY |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69309 | UBIQP_AVEFA |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CG83 | UBIQP_HORVU |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69315 | UBIQP_LINUS |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69322 | UBIQP_PEA |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69325 | UBIQP_SOYBN |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69310 | UBIQ_AVESA |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69313 | UBIQ_HELAN |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69317 | UBIQ_LUPPO |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P69326 | UBIQ_WHEAT |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q8H159 | UBQ10_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH33 | UBQ11_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q3E7T8 | UBQ14_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q1EC66 | UBQ3_ARATH |
| 97.4 | B | Ubiquitin-binding protein CUE2 | Q58G87 | UBQ3_ORYSJ |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P0CH32 | UBQ4_ARATH |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P69061 | RS27A_KLULA |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P05759 | RS31_YEAST |
| 97.4 | B | Ubiquitin-binding protein CUE2 | P42740 | UBIQP_AGLNE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P42739 | UBIQP_ACEPE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | Q3E7K8 | UBQ12_ARATH |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CH11 | RL401_CHLRE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CH10 | RL403_CHLRE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P49632 | RL40_CAEEL |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG71 | UBIQ1_CAEEL |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P59669 | UBIQP_GEOCY |
| 94.7 | B | Ubiquitin-binding protein CUE2 | Q9FHQ6 | UBQ9_ARATH |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P49633 | RL40_ACACA |
| 98.7 | B | Ubiquitin-binding protein CUE2 | P14799 | RS27A_NEUCR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P63048 | RL40_BOVIN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P63050 | RL40_CANLF |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P18101 | RL40_DROME |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P63052 | RL40_FELCA |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62987 | RL40_HUMAN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0C273 | RL40_MACFA |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62984 | RL40_MOUSE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P68205 | RL40_OPHHA |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P63053 | RL40_PIG |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0C275 | RL40_PONPY |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62986 | RL40_RAT |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0C276 | RL40_SHEEP |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P15357 | RS27A_DROME |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P29504 | RS27A_MANSE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P68202 | RS27A_PLUXY |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P68203 | RS27A_SPOFR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG53 | UBB_BOVIN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG54 | UBB_CAVPO |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG62 | UBB_CHICK |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG67 | UBB_GORGO |
| 96.1 | B | Ubiquitin-binding protein CUE2 | Q8MKD1 | UBB_HORSE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG47 | UBB_HUMAN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG49 | UBB_MOUSE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG65 | UBB_PANTR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG60 | UBB_PONPY |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG51 | UBB_RAT |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG55 | UBB_SHEEP |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CH28 | UBC_BOVIN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG66 | UBC_GORGO |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG48 | UBC_HUMAN |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG50 | UBC_MOUSE |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG64 | UBC_PANTR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG68 | UBC_PIG |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG61 | UBC_PONPY |
| 96.1 | B | Ubiquitin-binding protein CUE2 | Q63429 | UBC_RAT |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62976 | UBIQP_CRIGR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P0CG69 | UBIQP_DROME |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62972 | UBIQP_XENLA |
| 96.1 | B | Ubiquitin-binding protein CUE2 | Q865C5 | UBIQ_CAMDR |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P68197 | UBIQ_CERCA |
| 96.1 | B | Ubiquitin-binding protein CUE2 | P62975 | UBIQ_RABIT |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P33190 | RL40_TETPY |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0DJ25 | RL40_TETTS |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0C032 | RUB3_ORYSJ |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P23324 | UBIQP_EUPEU |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG82 | UBIQP_TETPY |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CH27 | RL402_TRYCR |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P46575 | RL40_EIMBO |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P14795 | RL40_TRYCR |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P14794 | RL40_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG76 | UBIQA_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG77 | UBIQD_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG78 | UBIQF_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG79 | UBIQG_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG81 | UBIQH_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG80 | UBIQI_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P0CG88 | UBIQJ_DICDI |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P22589 | UBIQP_PHYIN |
| 94.7 | B | Ubiquitin-binding protein CUE2 | P23398 | UBIQP_STRPU |
| 94.7 | B | Ubiquitin-binding protein CUE2 | Q8SWD4 | UBIQ_ENCCU |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P62992 | RS27A_BOVIN |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P62978 | RS27A_CAVPO |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P79781 | RS27A_CHICK |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P62979 | RS27A_HUMAN |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P68200 | RS27A_ICTPU |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P62983 | RS27A_MOUSE |
| 96.0 | B | Ubiquitin-binding protein CUE2 | P62982 | RS27A_RAT |
| 93.4 | B | Ubiquitin-binding protein CUE2 | P69201 | RL40_LEIMA |
| 92.1 | B | Ubiquitin-binding protein CUE2 | P21899 | RL40_TRYBB |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0C016 | RS27A_SCHPO |
| 100.0 | B | Ubiquitin-binding protein CUE2 | P0C8R3 | RS27B_SCHPO |
| 97.2 | B | Ubiquitin-binding protein CUE2 | Q9ARZ9 | R27AA_ORYSJ |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P59232 | R27AB_ARATH |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P51431 | R27AB_ORYSJ |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P59233 | R27AC_ARATH |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P0CG86 | RS271_HORVU |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P0CG87 | RS272_HORVU |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P47905 | RS27A_LUPAL |
| 97.2 | B | Ubiquitin-binding protein CUE2 | P27923 | RS27A_MAIZE |
| 93.2 | B | Ubiquitin-binding protein CUE2 | P14797 | RS27A_DICDI |
| 90.8 | B | Ubiquitin-binding protein CUE2 | P69200 | RL40_LEITA |
| 97.1 | B | Ubiquitin-binding protein CUE2 | P59272 | RS27A_DAUCA |
| 90.8 | B | Ubiquitin-binding protein CUE2 | Q39256 | UBQ8_ARATH |
| 95.2 | B | Ubiquitin-binding protein CUE2 | P84589 | UBIQ_LUMTE |
| 97.5 | B | Ubiquitin-binding protein CUE2 | P31753 | RS27A_ASPOF |